It is the integration of both redox signaling and the intracellular redox state that determines cellular response by coordinating multiple signaling networks. The long-term goal of our research program is to understand how NADPH oxidases can be manipulated to treat cardiovascular diseases. We have reported that Nox4 NADPH oxidase modifies diverse cellular responses. However, the molecular mechanisms by which Nox4 regulate cellular functions are poorly understood. Nox4 is expressed in cellular organelles whose functions are susceptible to changes in the redox potential. In contrast to the other Nox homologs, it is unlikely that Nox4-derived reactive oxygen species function as second messengers since 1) Nox4 is constitutively active; 2) activation of Nox4 is primarily regulated by its expression level; and 3) the reaction between thiols and H2O2 (the primary product of Nox4) is too slow to be of biological relevance. In pilot studies, we made the novel observations that 1) microRNA-9 (miR-9) is a novel regulator of Nox4 expression; 2) miR-25 induces expression of miR-9; 3) miR-9 elicits changes in Nox4 mRNA splicing; 4) miR-25 and miR-9 levels increase following vascular injury; and 5) changes in Nox4 levels modify the cellular glutathione redox potential and protein thiol status. We therefore hypothesize that miRNA-mediated changes in Nox4 expression dynamically regulate the cellular redox state and coordinate the expression of genes implicated in the development of vascular disease. Aim 1 will test the hypothesis that miR-induced miR expression mediates changes in Nox4 levels, cellular localization, and SMC activation. We will first determine the mechanism by which both miR-25 and miR-9 are required for decreasing Nox4 protein levels. We will then test the hypothesis that miR-9 alters the splice isoform profile and subcellular distribution of Nox4. Next, we will explore the mechanism by which miR-25 induces miR-9 expression. Finally, we will determine whether miR-9 modifies vascular smooth muscle cell (SMC) differentiation and the vascular response to injury. Aim 2 will test the hypothesis that Nox4 dynamically regulates the cellular redox state and transcriptional activity in cultured SMC and in the vessel wall. Proposed studies will determine the effect of changes in Nox4 expression on cellular thiol-disulfide status, the redox potential in different cellular compartments, and on the transcription of redox-sensitive genes. In vivo studies will explore how changes in SMC Nox4 modify vascular redox state and neointimal hyperplasia. This proposal is innovative in that we introduce a new paradigm whereby Nox4 acts as a redox rheostat, establishing the thiol oxidation state of cellular proteins to coordinate signaling event. These data may provide a unifying mechanism for the diverse and ambiguous functions of Nox4 across multiple biologic systems. Our data will be the first to provide evidence that one miRNA is indirectly induced by another miRNA. Finally, our findings will provide crucial insights into whether Nox4, miR-9, and miR-25 are therapeutic targets in vascular disease.